Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

Androgen receptor-dependent and independent functions of ARIP4

Androgen receptor (AR) is necessary for normal male phenotype development and essential for spermatogenesis. AR is a classical steroid receptor mediating actions of male sex steroids testosterone and 5-alpha-dihydrotestosterone. Numerous coregulators interact with the receptor and regulate AR activity on target genes. This study deals with the characterization of androgen receptor-interacting protein 4 (ARIP4). ARIP4 binds DNA, interacts with AR in vitro and in cultured yeast and mammalian cells, and modulates AR-dependent transactivation. ARIP4 is an active DNA-dependent ATPase, and this enzymatic activity is essential for the ability of ARIP4 to modulate AR function. On the basis of sequence homology in its ATPase domain, ARIP4 belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA repair, and homologous recombination. Similar to its closest homologs ATRX and Rad54, ARIP4 does not seem to be a classical chromatin remodeling protein in that it does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. However, ARIP4 is able to generate superhelical torsion on linear DNA fragments. ARIP4 is covalently modified by SUMO-1, and mutation of six potential SUMO attachment sites abolishes the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function. ARIP4 expression starts in early embryonic development. In mouse embryo ARIP4 is present mainly in the neural tube and limb buds. In adult mouse tissues ARIP4 expression is virtually ubiquitous. In mouse testis ARIP4 is expressed in the nuclei of Sertoli cells in a stage-dependent manner. ARIP4 is also present in the nuclei of Leydig cells, spermatogonia, pachytene and diplotene spermatocytes. Testicular expression pattern of ARIP4 does not differ significantly in wild-type, FSHRKO, and LuRKO mice. In the testis of hpg mice, ARIP4 is found mainly in interstitial cells and has very low, if any, expression in Sertoli and germ cells. Heterozygous Arip4+/ mice are fertile and appear normal; however, they are haploinsufficient with regard to androgen action in Sertoli cells. In contrast, Arip4 / embryos are not viable. They have significantly reduced body size at E9.5 and die by E11.5. Compared to wild-type littermates, Arip4 / embryos possess a higher percentage of apoptotic cells at E9.5 and E10.5. Fibroblasts derived from Arip4 / embryos cease growing after 2-3 passages and exhibit a significantly increased apoptosis and decreased proliferation rate than cells from wild-type embryos. Our findings demonstrate that ARIP4 plays an essential role in mouse embryonic development. In addition, testicular expression and AR coregulatory activity of ARIP4 suggest a role of ARIP4-AR interaction in the somatic cells of the testis.

Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Freedom ride... I have a dream

A play in two parts on the subject of racism. Part One presents the story of the Montgomery bus boycott arising from the refusal of Rosa Parkes to give up her seat on a bus and leading to the rise of Martin Luther King, Jr. Part Two is set in contemporary Australia, in the household of a group of young people who re-create the sixties' Freedom Ride in order to heal the spirit of Auntie, an Aboriginal woman, who is the centre of their world.

Of tolerance in mice and men : studies in APECED and Aire

Autoimmune diseases affect 5 % of the population and come in many forms, such as diabetes, rheumatoid arthritis and MS. However, how and why autoimmune diseases arise are not yet fully resolved. In this thesis, the onset of autoimmunity was investigated using both patient samples and a mouse model of autoimmunity. Autoimmune diseases are usually complex, due to a number of different causative genes and environmental factors. However, a few monogenic autoimmune diseases have been described, which are caused by mutations in only one gene per disease. One of such disease is called APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) and is enriched in the Finnish population. The causative gene behind APECED is named AIRE from AutoImmune REgulator. How malfunction of just one gene product can cause the multitude of disease components found in APECED is not yet resolved. This thesis sought out to find out more about the functions of AIRE, in order to reveal why APECED and other autoimmune diseases arise and what goes wrong? Usually, immune cells are taught to distinguish between self and non-self during their development. That way, immune cells can fight off bacteria and microbes while leaving the tissues and organs of the host organism itself unharmed. In APECED, the development of immune cells called αβ T cells is incomplete. The cells are not able to fully distinguish between self and non-self. This leads to autodestruction of self tissues and autoimmune disease. One of the achievements of this thesis was the finding that the development of another set of T cells called γδ T cells is not affected by AIRE in mice or in men. Instead, we found that another type of immune cell important in tolerance, called the dendritic cell is defective in APECED patients and is not able to respond to microbial stimulus in a normal fashion. Finally, we studied Aire-deficient mice and found that autoantibodies expressed in the mice were not targeted against the same molecules as those found in APECED patients. This indicates differences in the autoimmune pathology in mice and men. More work is still required before we understand the mechanisms of tolerance and autoimmunity well enough to be able to cure APECED, let alone the more complex autoimmune diseases. Yet altogether, the findings of this thesis work bring us one step closer to finding out why and how APECED and common autoimmune diseases arise.

The Pharmacology of Oxycodone : Studies In Vitro, In Vivo and In Humans

The antinociceptive properties of oxycodone and its metabolites were studied in models of thermal and mechanical nociception and in the spinal nerve ligation (SNL) model of neuropathic pain in rats. Oxycodone induced potent antinociception after subcutaneous (s.c.) administration in all models of nociception used in rats compared with morphine, methadone and its enantiomers. In the SNL model of neuropathic pain in rats, oxycodone produced dose dependent antinociception after s.c. administration. The antinociceptive effects of s.c. oxycodone were antagonized by naloxone but not by nor-binaltorphimine (Nor-BNI) a selective κ-opioid receptor antagonist indicating that the antinociceptive properties of oxycodone are predominantly μ-opioid receptor-mediated. The antinociceptive activity of oxymorphone, noroxycodone, and noroxymorphone, oxidative metabolites of oxycodone, were studied to determine their role in the oxycodone-induced antinociception in the rat. Of the metabolites of oxycodone s.c. administration of oxymorphone produced potent thermal and mechanical antinociception. Noroxycodone had a poor antinociceptive effect and noroxymorphone was inactive. Oxycodone produced naloxone-reversible antinociception after intrathecal (i.t) administration with a poor potency compared with morphine and oxymorphone. This seems to be related to the low efficacy and potency of oxycodone to stimulate μ-opioid receptor activation in the spinal cord in μ-opioid receptor agonist-stimulated (GTP)γ[S] autoradiography, compared with morphine and oxymorphone. All metabolites studied were more potent than oxycodone after i.t. administration. I.t. noroxymorphone induced a significantly longer lasting antinociceptive effect compared with the other drugs studied. The role of cytochrome P450 (CYP) 2D6-mediated metabolites on the analgesic activity of oxycodone in humans was studied by blocking the CYP2D6-mediated metabolism of oxycodone with paroxetine. Paroxetine co-administration had no effect on the analgesic effect of oxycodone compared with placebo in chronic pain patients, indicating that oxycodone-induced analgesia and adverse-effects are not dependent of the CYP2D6-mediated metabolism in humans. Although oxycodone has many pharmacologically active metabolites, they seem to have an insignificant role in oxycodone-induced antinociception in humans and rats.

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.

Milk casein-derived tripeptides : A special focus on blood pressure and vascular function

Increased consumption of low-fat milk products is inversely associated with the risk of hypertension. The beneficial effect of milk on blood pressure is attributed to high calcium and potassium content but also to specific peptide sequences, which are cleaved from milk protein during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or enzymatic hydrolysis. Milk products fermented with Lactobacillus helveticus contain casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro), which have been shown to possess antihypertensive effects in humans and in experimental animals. The aim of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. Another aim was to characterize the acute effects of tripeptides on blood pressure and arterial stiffness in mildly hypertensive humans. Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them attenuated the development of hypertension in two experimental models of hypertension, spontaneously hypertensive rat (SHR) and type 2 diabetic Goto-Kakizaki (GK) rat fed with high-salt diet. Significant differences in systolic blood pressure (SBP) were seen after 8 weeks treatment with tripeptide-containing products compared to control product. Plant sterols did not enhance this effect. Two differently produced tripeptide powders produced a similar attenuating effect on SBP in SHR. In mildly hypertensive subjects, a single administration of tripeptide- and plant sterol-containing fermented milk product decreased both SBP and diastolic blood pressure (DBP) over a period of 8 hours. Protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the renin-angiotensin system (RAS) in SHR and GK. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOx and citrulline suggest increased nitric oxide (NO) production by the tripeptides. Taken together, Ile-Pro-Pro and Val-Pro-Pro -containing products attenuate the development of hypertension after long-term treatment in experimental models of hypertension and possess an acute antihypertensive effect in mildly hypertensive subjects. In addition, these tripeptides show endothelium-mediated beneficial effects on vascular function. Attenuation of blood pressure increase by the tripeptides in experimental animals involves RAS, but its role in the antihypertensive effect in humans remains to be elucidated.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Studies on the pro-oxidative properties and neurotoxic potential of Angeli's salt-derived nitroxyl (HNO/NO-)

The biological function of nitric oxide and its oxidized forms has received a great deal of attention over the past two decades. However much less attention has been focused on the reduced nitric oxide, nitroxyl (HNO). Unlike NO, HNO is highly reactive species and thus it needs to be generated by using donor compounds under experimental conditions. Currently there is only one donor available, Angeli s salt, which releases HNO in a controlled fashion under pysiological conditions. Prior studies have shown the pro-oxidative and cytotoxic potential of Angeli s salt compared to NO donors. The high reactivity of HNO with cysteine thiols is considered to form the biochemical basis for its unique properties compared to other nitrogen oxides. Such thiol modification cold result in disturbances of vital cellular functions and subsequently to death of disturbance sensitive cells, such as neurons. Therefore modification of proteins and lipids was studied in vitro and the potential neurotoxicity was studied in vivo by local infusion of Angeli s salt into the rat central nervous system. The results show that under aerobic in vitro conditions, HNO can, subsequent to autoxidation, cause irreversible oxidative modification of proteins and lipids. These effects are not however seen in cell culture or following infusion of Angeli s salt directly into the rat central nervous tissue likely due to presence of lower oxygen and higher thiol concentration. However, due to high reactivity with thiols, HNO can cause irreversible inactivation of cysteine modification sensitive enzymes such as cysteine proteases papain in vitro and cathepsin B in cell culture. Furthermore it was shown that infusion of HNO releasing Angeli s salt into the rat central nervous system causes necrotic cell death and motor dysfunction following infusion into the lumbal intrathecal space. In conclusion, the acute neurotoxic potential of Angeli s salt was shown to be relatively low, but still higher compared to NO donors. HNO was shown to affect numerous cellular processes which could result in neurotoxicity if HNO was produced in vivo.

p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.